1. Field of the Invention
The present invention relates to high throughput methods for screening for optimal solution conditions for crystallizing proteins.
2. Related Art
Advances in X-ray crystallography have provided three-dimensional structures of thousands of proteins. In spite of these advances, protein aggregation continues to be a common problem that can lead to unsuccessful crystallization of proteins. This problem is becoming more prominent in attempts at crystallizing many different proteins and protein complexes in a structural genomics scale.
At the Berkeley Structural Genomics Center (BSGC), a purified protein sample is obtained after one or more chromatography steps (immobilized metal affinity chromatography (IMAC), ion-exchange chromatography, and size exclusion chromatography), and the sample is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1971) to determine the chemical purity of the protein. In the process of developing techniques to automate protein purification, one tries as much as possible to use a set of generic buffers. Very little is known about the properties of the proteins a priori, except for theoretical pI, molecular weight and amino acid composition. The general practice is to use one or two favorite buffers where pH and salt concentration are some of the variables. However, a protein has complex properties and its condition and behavior depend very much on the environment surrounding it. In the past when faced with difficulties of aggregation and precipitation, one would try to change purification parameters, add or remove fusion tags, test some additives (e.g., DTT, glycerol, etc.). After purification, the protein is concentrated and the presence of aggregates is assessed using for example, the dynamic light scattering method. This method as described by Zulauf & D'Arcy (1992) has shown that the presence of aggregates in the protein solution may inhibit crystal nucleation or growth (Habel et al., 2001, Ferre-D'Amare& Burley, 1997).
In order to grow crystals, one must identify the conditions under which proteins will precipitate out of solution. The technique of vapor diffusion is commonly used to analyze this controlled precipitation and by using a sparse matrix approach (Jancarik and Kim, 1991 and further expanded by Hampton Research (Aliso Viejo, Calif.)), one can test a large number of crystallization conditions. This assumes that the starting protein solution is not aggregated or precipitated. In both the preparation for NMR or X-ray crystallography samples, one must start with a protein solution that is homogeneous and monodisperse. Lepre and Moore (1998) developed a modified vapor diffusion method to efficiently screen solvent conditions for NMR samples in order to optimize solubility. Collins et al. have developed preliminary solubility screens using different buffer components and focus on the combination of best anion and cation buffers for solubility improvement (Acta Cryst. (2004) D60, 1674-1678).